Example 5
Using N-succinimidyl-3-(4-hydroxy-3-[125I]iodophenyl)propionate, Bolton-Hunter Reagent (NEX120, PerkinElmer), the purified ST03-Cupid protein was radio-iodinated. Specifically, Bolton-Hunter Reagent in an amount of 20 times the number of moles of the used protein was taken in an Eppendorf tube, the solvent was then vaporized, and the protein solution was then added thereto. The mixture was reacted on ice for 2 hours. Thereafter, the reaction mixture was subjected to a desalination column (PD MiniTrap G-25, GE Healthcare) to remove unreacted radio-iodination Bolton-Hunter Reagent, and the resultant was then subjected to an ultrafiltration column (VIVASPIN Turbo 4, Sartorius) for concentration. After completion of the concentration, the amount of radioactivity was measured using a dose calibrator (CRC-25w, CAPINTEC), and the quality of the protein was checked by performing CBB staining according to SDS-PAGE.
